It has been long been known that eukaryotic ribosomes are heterogeneous in nature. Ribosome heterogeneity is exemplified by the differential presence of certain ribosomal proteins and ribosomal RNAs, modification of ribosomal proteins and RNA, and association of non-ribosomal proteins with ribosomal particles. However, it remains unknown whether the heterogeneous pools of ribosomes reflect distinct activities in protein biosynthesis. More recently, it has become clear that the internal ribosome entry sites (IRES) located in certain viral mRNAs can bind directly and with high affinity to mammalian 40S subunits. The aims of this proposal are to examine whether functionally heterogeneous populations of ribosomes exist in mammalian cells and whether distinct ribosome populations are recruited to picornaviral and hepatitis C viral IRES elements in infected cells. In the first aim, the composition and activity of total, unbound and polysome-bound ribosomal subunits from uninfected cells will be compared to those isolated from picornavirus infected cells by electrospray mass spectrometry (ES-MS). In the second aim, the presence of altered ribosomes in viral mRNA-complexes will be examined. Specifically, thiouridine-containing viral RNA will be generated in HeLa cells expressing the Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) enzyme. UPRT will convert thiouracil to thiouridine 5'monophosphate nucleotides whose triphosphates can be selectively incorporated into viral RNA by the viral RNAdependent RNA polymerase in the presence of actinomycin D. Polysomal thio-labeled RNA will be biotinylated, isolated by streptavidin chromatography and associated ribosomes characterized by ES-MS. This method will also be used to identify IRES-binding proteins that are tightly linked to the viral RNA in infected cells. Finally, the roles of modified ribosomes in various steps of translation initiation will be examined in reconstituted translation systems. The outcome from these studies will reveal roles of modified ribosomes in mammalian cells and will provide insights by which IRES elements recruit host cell ribosomes during viral infection, revealing new potential targets for antiviral therapeutics.